Nanoparticle isoflavone compositions &amp; methods of making and using the same

ABSTRACT

The present invention is directed to formulations of genistein and methods for making and using the same. In particular embodiments, the formulations described herein include suspension formulations of nanoparticulate genistein.

TECHNICAL FIELD

The present invention relates to compositions including genistein andmethods for producing and utilizing such compositions.

BACKGROUND

Genistein is a pharmaceutically active isoflavone. In the body,genistein interacts with various enzymes that have wide-ranging actionsin many tissues. Therefore, the potential therapeutic impacts ofgenistein are diverse. However, genistein has proven difficult toformulate and deliver to subjects in a manner that achieves andmaintains therapeutically effective blood plasma levels.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the thirty-day survival rates in groups of mice receiving asolution formulation of genistein versus the thirty-day survival ratesin groups of mice receiving a genistein suspension formulation asdescribed herein.

FIG. 2 shows the thirty-day survival rates of mice after subcutaneousadministration of a genistein suspension formulation as described hereinat 24, 18, 12 , or 6 hr pre-irradiation.

FIG. 3 shows the thirty-day survival rates of mice after subcutaneousand intramuscular administration of a genistein suspension formulationas described herein.

FIG. 4 shows the free genistein concentration achieved after oraladmininstration of a genistein suspenstion formulation as describedherein versus that achieved by a solution formulation of genistein.

FIG. 5 shows the total genistein concentration achieved after oraladmininstration of a genistein suspenstion formulation as describedherein versus that achieved by a solution formulation of genistein.

FIG. 6 shows the free genistein concentration achieved after oraladmininstration of a genistein suspenstion formulation as describedherein versus that achieved by a non-nanoparticulate suspensionformulation of genistein.

FIG. 7 shows the total genistein concentration achieved after oraladmininstration of a genistein suspenstion formulation as describedherein versus that achieved by a non-nanoparticulate suspensionformulation of genistein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Compositions of genistein compounds are described herein. In particularembodiments, the compositions described herein are pharmaceuticalformulations suitable for oral or parenteral administration. Generally,given the desired therapeutic applications for genistein, it isdesirable to deliver genistein to a subject in a manner that (i)achieves a therapeutic blood plasma concentration in a relatively shortperiod of time and (ii) maintains a therapeutic blood plasmaconcentration over an extended period of time. Using availableformulations of genistein, however, it has been found that relativelyhigh doses of genistein are often required to achieve and maintaintherapeutic blood plasma concentrations of genistein. This isparticularly true when genistein is administered orally. However, evenwhen genistein is formulated for parenteral administration (e.g., viaintravenous injection or infusion. Intravascular injection, subcutaneousinjection, or intramuscular injection), it is often the case thatrelatively large volumes of drug formulation must be delivered in orderto achieve and maintain therapeutic blood plasma concentrations.

Genistein is practically insoluble in water, requiring greater than50,000 parts water at 25° C. to dissolve one part genistein.Furthermore, when delivered orally, genistein has shown poorbioavailability, which may be due, at least in part, to the compound'slow water solubility. Therefore. In light of genistein's generally lowbioavailability and water solubility, achieving and maintainingtherapeutic blood plasma concentrations of genistein can require highdoses of genistein delivered at relatively high dose frequencies.Genistein compositions with high concentrations of genistein thatprovide significantly increased bioavailablity are described herein.Moreover. In particular embodiments, the genistein compositionsdescribed herein maintain therapeutic blood plasma levels of genisteinover an extended period of time.

The genistein formulations described herein are suitable for oral andparenteral administration. Additionally, the formulations describedherein potentially provide several advantages. For example, because theycan be used to achieve therapeutic plasma concentrations of genisteinusing less amounts of drug substance and. In some embodiments,relatively fewer doses, the formulations described herein may reduce thecosts of genistein treatments as well as any potential side effects thatmay be associated with relatively higher doses of the compound.Moreover, because the formulations described herein enable delivery oftherapeutic amounts of genistein using relatively smaller administeredamounts of formulated drug, they may ease patient compliance and expandthe contexts in which administration of genistein may be utilized.

The formulations described herein also exhibit desirable stabilitycharacteristics, are scalable for commercial production and. In specificembodiments, may increase the circulating half-life of genistein afteradministration.

Methods of treating subjects at risk for or suffering from variousdiseases and disorders suitable for treatment using genistein are alsodescribed herein.

I. Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “abuffer” includes a plurality of such buffers, reference to “the buffer”is a reference to one or more buffers and equivalents thereof known tothose skilled in the art, and so forth.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

As used herein, “nanoparticulate” refers to material exhibiting a volumediameter, as measured using laser light diffraction, wherein the D(0.50) of the material is 0.5 μm or less and no particles measuregreater then 2 μm. Particle size analysis using laser light diffractionis a technique based on light being scattered through various angleswhich are directly related to the size of the particles. By measuringthe angles of light scattered by the particles being analyzed and theintensity of this scattered light, a particle size distribution can becalculated. Techniques for use in analyzing particle size in the contextof the present disclosure can be referred to as static light scattering,Rayleigh light scattering, low angle light scattering (LALS), multipleangle light scattering (MALS) Fraunhofer diffraction, or Mie Scattering.Measurement of particle size distributions using Mie Scattering allowsfor the determination of particle size distributions through thedirection measurement of mass.

Two theoretical applications to the analysis of particle size by laserlight diffraction are based on assumptions about the properties of theparticles. Fraunhofer theory considers the following: particles arespherical, non-porous and opaque; particle diameters are greater thanthe wavelength of the laser light used in the analysis; and particlesare distant enough from each other not to interfere in the diffractionof light, exhibit random motion, and diffract light with the sameefficiency regardless of size and shape. Mie theory considers thedifferences in refractive index between the particles and the suspendingmedium, which allows the measurement technique to account for particlesin the size range below the wavelength of the laser light used in theanalysis. The relative amounts of different size particles aredetermined by measuring the intensity of light scattered at differentangles. As the particles get close to or smaller than the wavelength oflight, more of the light intensity is scattered to higher angles andback-scattered, Mie Scattering Theory accounts for this differentbehavior. In order to make particle size measurements, the lightintensity pattern is measured over the full angular range. When theparticle size is larger than the wavelength of the incident light, theMie equation reduces to the Fraunhofer equation. An array of detectors.Including high-angle and back-scatter detectors, and multiple lightsources of different wavelengths are typically employed to allowmeasurement of the full size range in one analysis. Equipment suited foruse in analyzing particle size by laser light diffraction iscommercially available and manufactured, for example, by HoribaInstruments, Irvine, Calif.

The term “volume diameter” as used herein refers to the size of aparticle as measured using a laser diffraction particle size analyzer,operating in the Mie Scattering Theory diffraction mode, equipped with asuspension dispersion sample chamber (e.g., as available from HoribaInstruments, Irvine, Calif., USA). For purposes of the presentdescription, volume diameter is given as a particle size distributiondefined by one or more of D (0.10), D (0.50) and D (0.90). When referredto herein, the term D (0.10) indicates the volume frequency distributionof particles for which 10% of the sample is below the referenced size,the term D (0.50) indicates the volume frequency distribution ofparticles for which 50% of the sample is below the referenced size, andthe term D (0.90) indicates the volume frequency distribution ofparticles for which 90% of the sample is below the referenced size.

The term “parenteral” as used herein refers to delivery of an activeagent or formulation to a subject via any route or means other than oraladministration. For example, for purposes of the present disclosure,parenteral formulations include formulations and systems for topical,transdermal, and buccal delivery. The term “parenteral” as contemplatedherein further encompasses delivery via suppository and compositionssuited to formulation as a suppository. For purposes of the presentdisclosure, the term “parenteral” additionally emcompasses delivery viainfusion or injection, such as, for example. Intravenous injection.Intravenous infusion, intravascular injection, subcutaneous injection,and intramuscular injection.

As used herein, “pharmaceutical composition” refers to a compositionthat includes genistein in combination with one or more pharmaceuticallyacceptable excipients or adjuvants and is suitable for oral orparenteral administration to a subject.

The term “radioprotective agent” refers to agents that protect cells orliving organisms from the deleterious cellular effects that result fromexposure to ionizing radiation. These deleterious cellular effectsinclude damage to cellular DNA, such as DNA strand break, disruption incellular function, cell death and/or carcinogenesis. More particularly,the hematopoietic system is a rapidly dividing system and is thereforecentrally affected by exposure to high-dose whole body ionizingradiation. Bone marrow aplasia and the resultant leukopenia,erythropenia and thrombocytopenia predispose the animal or human toinfection, hemorrhage and ultimately death. For purposes of the presentdisclosure, a radioprotective agent may be one that is administeredprophylactically prior to potential radiation exposure, with suchadministration resulting in the prevention, reduction in severity, orslowing of the symptoms or effects of exposure to ionizing radiation,should such an exposure occur. Additionally, a radioprotective agent maybe used as a treatment for radiation exposure, administered afterexposure to ionizing radiation has occurred, with such administrationresulting in mitigation (i.e., prevention, reduction in severity,slowing, halting, or reversal of symptoms or effects that are otherwiseassociated with exposure to a given dose of ionizing radiation).

A “subject” for purposes of this disclosure is an animal to which aformulation as described herein can be administered in order to achievea therapeutic effect. In one embodiment, the subject is a human being.

“Therapeutically effective” refers to an amount of genistein or anamount of a formulation of genistein as described herein which achievesa therapeutic effect by inhibiting a disease or disorder in a patient orby prophylactically inhibiting or preventing the onset of a disease ordisorder. A therapeutically effective amount may be an amount whichrelieves to some extent one or more symptoms of a disease or disorder ina patient; returns to normal either partially or completely one or morephysiological or biochemical parameters associated with or causative ofthe disease or disorder; and/or reduces the likelihood of the onset ofthe disease of disorder.

II. Genistein Formulations

Genistein is one of several known isoflavones that are normally found inplants The main sources of natural genistein are soybeans and otherlegumes. Genistein is commercially available and may be obtained insynthetic, purified form. Synthetic genistein is available, for example,as BONISTEIN from DSM Nutritional Products (DSM Nutritional Products.Inc. Parsippany, N.J.). Genistein's chemical name is5,7-dihydroxy-3-(4-hydroxyphenyl)-chromen-4-one (IUPAC). Genistein'schemical structure is shown as:

The genistein formulations described herein are suspension formulationsthat include nanoparticulate genistein suspended in a suspension mediumformed of one or more carriers, excipients, and/or diluents. Inparticular embodiments, the formulations are provided as pharmaceuticalcompositions, and the carriers, excipients and/or diluents used informing such compositions are selected from pharmaceutically acceptablematerials. Pharmaceutically acceptable carriers, excipients and diluentssuited for therapeutic use are well known in the pharmaceutical art, andare described, for example. In Remington's Pharmaceutical Sciences,Maack Publishing Co. (A. R. Gennaro (Ed.) 1985). In one such embodiment,the formulations as disclosed herein may include a suspension comprisingnanoparticulate genistein suspended within a suspension medium includinga water soluble polymer and a nonionic surfactant. The genistein used inthe formulations described herein may be naturally derived orsynthetically produced genistein. Pharmaceutical compositions ofgenistein as described herein can be formulated to be simultaneouslysuitable for both oral and parenteral administration. Though theformulations of genistein described herein are characterized assuspensions. In some embodiments, depending on the carriers, excipientsand diluents included in the medium within which the nanoparticulategenistein is suspended, a measureable amount of genistein may also bedissolved within the suspension medium.

Nanoparticulate genistein suitable for use in the formulations disclosedherein may be prepared according to known methods for producingnano-sized particles. In one embodiment, natural or synthetic genisteinmaterial may be nanomilled according to milling techniques known in theart. In one embodiment, nanomilling may include wet bead millingutilizing an agitator bead mill in a horizontal grinding container forcontinuous dispersion and fine wet grinding. In another embodiment,using a bead mill such as a DYNO-mill (CB Mills, Gurnee, Ill.), thenecessary energy for dispersion and grinding is transmitted to thegrinding beads through agitator discs mounted on an agitator shaft.

In one embodiment, a nanoparticulate genistein composition as describedherein is provided by introducing genistein suspended in apharmaceutically acceptable suspension medium into a bead mill. In suchan embodiment, the genistein suspension is fed into the bead mill andmilled in a manner that results in a pharmaceutical genisteincomposition characterized by nanoparticulate genistein suspended withinthe pharmaceutically acceptable suspension medium. In one suchembodiment, a genistein suspension formulation may be fed continuouslythrough the bead mill until a suspension composition containingnanoparticulate genistein material of a defined particle sizedistribution is reached. For example, the genistein formulation may benanomilled by recirculating the volume of the suspension through thebead mill, followed by one or more single passes through a bead mill toreach a pharmaceutical composition exhibiting the desired genisteinparticle size distribution. The particle size of the genistein materialsuspended within a pharmaceutical composition as described herein can becontrolled by adjusting the parameters of the bead mill and the grindingconditions. For example, the particle size produced by nanomillinggenistein or a genistein suspension formulation in a bead mill may becontrolled by bead size, bead load/suspension weight ratio, suspensioncomposition, agitation rate, and milling time.

Though nanomilling is generally referenced herein as a means forproducing nanoparticulate material suitable for use in the formulationsdescribed herein, the nanoparticulate material can be produced othersuitable techniques as well. For example, nanoparticulate genisteinmaterial as used herein can be produced using one or more known wetmilling techniques, super-critical or compressed fluid techniques, hotor high-pressure homogenization, emulsification techniques, evaporativeprecipitation, antisolvent precipitation, microprecipitation, cryogenictechniques, complexation techniques, ultrasonication techniques, orsolid dispersion techniques. Spray drying and lyophilization may be usedpost-processing to isolate nanoparticles resulting from an aqueous orsolvent dispersion technique.

The genistein included in the formulations described herein is ananoparticulate material as defined herein. In one embodiment, thecompositions disclosed herein may comprise nanoparticulate genisteinmaterial exhibiting a D (0.50) of 0.2 μm or less. In on such embodiment,the nanoparticulate genistein material exhibits a D (0.50) of 0.2 μm orless and a D (0.90) of 0.5 μm or less. In yet another embodiment, thenanoparticulate genistein material exhibits a Q (0.50) of 0.2 μm or lessand a D (0.90) of 0.4 μm or less.

The nanoparticulate genistein material included in the formulationsdescribed herein is suspended within a suspension medium that includesone or more carriers, excipients and/or diluents. As described herein.In particular embodiments, such carriers, excipients and diluents areselected from pharmaceutically acceptable materials to facilitatepreparation of pharmaceutical compositions that can be administered to asubject at risk for or suffering from a disease or disorder, such as,for example, a disease or disorder as described herein.

One or more nonionic surfactants may be included in the suspensionmedium to facilitate wetting and aid in preventing agglomeration of thenanoparticulate genistein drug substance. Nonionic surfactants suitablefor use in the formulations described herein may be selected from, forexample, polysorbates, poloxamers, polyoxyethylene castor oilderivatives (e.g., Cremophor EL, Cremophor RH60), bile salts, lecithin,12-Hydroxystearic acid-polyethylene glycol copolymer (e.g., Solutol HS15), and the like. In specific embodiments, the formulations describedherein include a nonionic surfactant selected from polysorbate 80 (Tween80), polysorbate 20 (Tween 20), Poloxamer 188, and combinations thereof.In one embodiment, the total nonionic surfactant content ranges fromabout 0.01% to about 10% by weight (w/w). In another embodiment, thetotal nonionic surfactant content ranges from about 0.1% to about 10%(w/w). In certain such embodiments, the total amount of nonionicsurfactant is selected from about 0.2% to about 5% (w/w), about 0.2% toabout 1% (w/w), about 0.2% to about 1% (w/w), about 0.2% to about 0.6%(w/w), and about 0.2% to about 0.8% (w/w).

The suspension formulations described herein may include one or morewater soluble polymers, which may serve, for example, to enhance theviscosity of the suspension or to stabilize nanoparticulate genisteinagainst particle agglomeration or potential deleterious effects fromother formulation components. Water soluble polymers arepharmaceutically acceptable polymers that can be dissolved or dispersedin water. Suitable water soluble polymers for use in the formulationsdescribed herein may be selected from, for example, vegetable gums, suchas alginates, pectin, guar gum, and xanthan gum, modified starches,polyvinyl pyrrolidone (PVP), hypromellose (HPMC), methylcellulose, andother cellulose derivatives, such as sodium carboxymethylcellulose,hydroxypropylcellulose, and the like. In certain embodiments, theformulations described herein may include a poloxamer, such as Poloxamer188, as a water soluble polymer. Poloxamer 188 is both a polymer andsurfactant. In other embodiments, the formulations described herein mayinclude Povidone K17 as a water soluble polymer. Where one or more watersoluble polymers are included in the suspension formulations describedherein. In specific embodiments, the total water soluble polymer contentranges from about 0.5% to about 15% (w/w). For example. In certain suchembodiments, the total water soluble polymer content ranges from about1% to about 10% (w/w). In other such embodiments, the total watersoluble polymer content may be selected from about 5% to about 15%(w/w), about 10% to about 15% (w/w), and 12% to about 15% (w/w), about1% to about 8% (w/w), and about 1% to about 5% (w/w).

In particular embodiments, the suspension medium included in thesuspension formulation includes a combination of one or more nonionicsurfactants with one or more water soluble polymers. Where that is thecase, the nonionic surfactant constituent and water soluble polymerconstituent can be selected from the materials already described herein.Including combinations of such materials. Moreover, where the suspensionmedium includes a combination of nonionic surfactant and water solublepolymer, the total nonionic surfactant and total water soluble polymerincluded in the suspension formulation can be selected from thoseamounts already detailed. For example, where the suspension mediumincludes a combination of nonionic surfactant and water soluble polymer,the total nonionic surfactant content may be selected from about 0.01%to about 10% (w/w), about 0.1% to about 10% (w/w), about 0.2% to about5% (w/w), about 0.2% to about 1% (w/w), about 0.2% to about 1% (w/w),about 0.2% to about 0.6% (w/w), and about 0.2% to about 0.8% (w/w), andthe total water soluble polymer content may be selected from about 0.5%to about 15% (w/w), about 1% to about 10% (w/w), about 5% to about 15%(w/w), about 10% to about 15% (w/w), about 12% to about 15% (w/w), about1% to about 8% (w/w), and about 1% to about 5% (w/w). In one suchembodiment, the nonionic surfactant constituent may be present in anamount ranging from about 0.1% to about 1% (w/w) and the water solublepolymer constituent may be present in an amount ranging from about 1% toabout 15% (w/w). In another such embodiment, the nonionic surfactantconstituent may be present in an amount ranging from about 0.2% to about1% (w/w) and the water soluble polymer constituent may be present in anamount ranging from about 5% to about 15% (w/w). In specific embodimentsof the suspension formulations where the suspension medium includes botha nonionic surfactant and a water soluble polymer, the nonionicsurfactant may be selected from a polysorbate, such as polysorbate 80(Tween 80) and polysorbate 20 (Tween 20), the water soluble polymer maybe selected from a poloxamer, such as Poloxamer 188, and a PVP, such asPovidone K17, with the nonionic surfactant and water soluble polymerbeing included in the formulation at any of the relative amountsdetailed herein.

The suspension medium included in the suspension formulations accordingto the present description may also include a carrier. For example,carriers suitable for use in the formulations described herein includepharmaceutically acceptable aqueous carriers such as, for example,sterile water, physiologically buffered saline Hank's solution, Ringer'ssolution, and any other suitable aqueous carrier. The suspensionformulations described herein can utilize buffers such as, for example,one or more of a citrate buffer, phosphate buffer, TRIS buffer, and aborate buffer to achieve a desired pH and osmolality. For example, thetypical pH range for formulating injectable pharmaceutical products isfrom about 2 to about 12. In some embodiments, the pH of the formulationmay fall in a range that more closely approximates physiologic pH. Forexample. In certain embodiments, the suspension formulations describedherein are formulated to exhibit a pH selected from a range of fromabout 4 to about 8 and a range of from about 5 to about 7.

The suspension formulations described herein can also include one ormore diluents. Suitable diluents may be selected from, for example,pharmaceutically acceptable buffers, solvents and surfactants.

Suspension formulations prepared as described herein are suited toproviding high concentration genistein formulations formulationscontaining genistein in amounts of about 250 mg/mL, or greater). Thoughgenistein exhibits low to virtually no solubility in severalpharmaceutically acceptable solvents, the nanoparticulate suspensionformulations described herein can incorporate genistein up to and over300 mg/ml. In specific embodiments, genistein formulations as describedherein may incorporate genistein in amounts ranging from about 250 mg/mLto about 500 mg/mL, In certain such embodiments, the amount of genisteinincluded in a suspension formulation as described herein may be selectedfrom about 200 mg/ml to about 400 mg/ml, from about 250 mg/ml to about350 mg/ml, and from about 275 mg/ml and about 325 mg/ml.

The relative amount of genistein included in the suspension formulationsdescribed herein may be varied, as desired, to achieve a formulationhaving a desired total content of genistein. For example, the suspensionformulations as described herein may include up to about 85% (WAN)genistein. In certain such embodiments, the relative amount of genisteinis selected from up to about 75% (w/w), up to about 65% (w/w), and up toabout 50% (w/w). Alternatively, embodiments of the suspensionformulations described herein may include an amount of genisteinselected from a range of about 40% to about 75% (w/w), a range of about40% to about 65% (w/w), a range of about 40% to about 50% (w/w), a rangeof about 50% to about 75% (w/w), and a range of about 50% to about 65%(w/w),

The inventors have also found that suspension compositions preparedaccording to the present description can increase bioavailability ofgenistein relative to solution formulations. In particular, as isillustrated in the experimental examples that follow, suspensionformulations prepared as described herein exhibited significantlyimproved relative bioavailability when compared to solution formulationsprepared using, for example, pharmaceutically acceptable PEG solvent.Such a result runs counter to what would be generally expected. Forexample. In certain embodiments, relative to a solution formulation ofgenistein or formulations of genistein incorporating larger sizedgenistein material, a suspension formulation as described hereinprovides an increase in peak total genistein serum concentrations of upto 300%. In particular such embodiments, the increase in peak totalgenistein serum concentration ranges from about 50% to about 300%. Inother such embodiments, the increase in peak total genistein serumconcentration is selected from about 50% or greater, about 75% orgreater, about 100% or greater, and about 200% or greater.

The combination of high drug loading and significantly increasedrelative bioavailability provided by formulations described hereinpresent several advantages, The significant jump in drug loading by thegenistein suspension formulations described herein facilitatesadministration of therapeutically effective amounts of genistein tosubjects in need thereof using much less formulated drug substance,which. In turn, can increase patient compliance and facilitatemanufacture of a genistein drug product that is much better suited toadministration of genistein in therapeutic contexts requiring deliveryof relatively high doses of genistein. Moreover, the increase inbioavailability afforded by the genistein suspension formulationsdescribed herein provides the added benefit of reducing the amount ofgenistein that must be delivered to a subject in order to achieve andmaintain therapeutic genistein blood plasma levels. Therefore, theformulations described herein offer a significant reduction in therelative amount of administered genistein required to achieve andmaintain a therapeutic benefit, which can reduce the costs of genisteintreatments, work to mitigate or avoid potential side effects that may beassociated with relatively higher doses of the compound, and furtherdecreases the amount of formulated drug substance required to achieveand maintain therapeutic efficacy.

Even further, the suspension formulations taught herein can beformulated such that that a single given formulation is suited to bothoral and parenteral delivery. Where a suspension formulation asdescribed herein is prepared for parenteral delivery it can bemanufactured according to standard methods to provide a sterilecomposition deliverable via, for example. Intravenous injection orinfusion. Intravascular injection, subcutaneous injection, orintramuscular injection. The suspension formulations described hereincan be prepared to exhibit viscosities suited for the desired route ofparenteral administration and can be manufactured and packaged in anymanner suited to the desired application. Including, for example, as aformulation deliverable via intravenous injection or infusion.Intravascular injection, subcutaneous injection, or intramuscularinjection. In certain embodiments, the formulations described herein maybe included in pre-filled syringes prepared for administration of agiven dose or range of doses of genistein.

Where prepared for oral administration, the formulations may be preparedin any suitable manner and using any suitable devices for oraladministration of desired doses of genistein. For example, when theformulations described herein are prepared for oral delivery, they maybe prepared as a liquid suspension that can be metered to deliver adesired dose or incorporated into capsules, such as gelatin or softcapsules, suited for delivery of liquid formulations. Alternatively,formulations as described herein prepared for oral administration can beloaded into prefilled sachets or premetered dosing cups. Genisteinformulations prepared for oral administration may optionally include oneor more pharmaceutically acceptable sweetening agents, preservatives,dyestuffs, flavorings, or any combination thereof.

III. Methods

The genistein suspension formulations described herein can be used totreat subjects suffering from or at risk for a disease or disordertreatable with genistein. Clinical trials, animal studies, cell-cultureexperiments, and epidemiological studies have provided evidence thatgenistein exerts various physiological effects. Examples of diseases anddisorders amenable to treatment by genistein are described herein.However, the potential therapeutic applications of genistein are notlimited to those described herein, and genistein formulations accordingto the present description can be used to treat a subject at risk for orsuffering from any disease or disorder for which administration ofgenistein will be therapeutically effective.

As one example, genistein has displayed antitumor, antimetastatic andantiangiogenic (suppression of blood-vessel growth) properties in tissueculture and in vivo. Several epidemiological studies suggest thatsoybean consumption may contribute to lower incidence of breast, colon,prostate, thyroid, and head and neck cancers—an effect that isattributed to genistein and other isoflavones (Takimoto et al., CancerEpidemiol Biomarkers Prey. 2003 November 12(11 Pt 1): 1213-21; Wei etat., J Nutr. 2003 November 133(11 Suppl 1): 3811S-3819S; Sakar, F. H.and Y. Li, Cancer Invest. 2003; 21(5): 744-57; Magee P. J. and I. R.Roland, Br J Nutr. 2004 April; 91(4): 513-31; Park, O. J. and Y. J.Surh, Toxicol Lett. 2004 Apr. 15; 150(1): 43-56; Messina. M. J., NutrRe. 2003 April 61(4): 117-31). Genistein has also been reported toinhibit non-Hodgkin's lymphoma, melanoma, lung cancers, and ovariancancer (Wei et al. 2003; Mohammad et at, Mol Cancer Ther. 2003 December2(12): 1361-8; Nicosia et at, Hematol Oncol Ciin North Am. 2003 August17(4): 927-43; Sun et at, Nutr Cancer. 2001; 39(1): 85-95). Tissueculture experiments suggest that genistein's cancer-fighting effectsoccur at dosages that are hard to attain from food alone, unless oneeats very large amounts of soy products. Reliable genistein dosingtherefore requires the use of concentrated supplements (Magee and Roland2004).

The genistein formulations may, therefore, be used in methods ofinhibiting the onset, development or progression of certain cancers,such as cancers selected from breast, colon, prostate, thyroid, and headand neck cancers. In one such embodiment, a subject at risk fordeveloping a breast, colon, prostate, thyroid, head or neck cancer isidentified and a therapeutically effective amount of a genisteinformulation selected from any of those described herein is administeredto the subject. The genistein formulations described herein may also beused in methods of treating cancer. In a particular embodiment, apatient at risk for or suffering from a cancer responsive to genisteintreatment, such as for example, a cancer selected from non-Hodgkin'slymphoma, melanoma, lung cancers, and ovarian cancer is identified and atherapeutically effective amount of a genistein formulation selectedfrom any of those described herein is administered to the subject.

The ability of genistein and related soy isoflavones to reducepost-menopausal bone-loss has also been shown in many studies. Thesesubstances prevent bone loss and promote bone formation, especially inthe spine. Among the dosage regimens found to be effective are: 1 mg/daygenistein+0.5 mg/day daidzein+42 mg/day other isoflavones (biochanin Aand formononetin. In this case); 54 mg/day genistein; 57 mg/dayisoflavones; 65 mg/day isoflavones; 90 mg/day isoflavones (Morabito etal. J Bone Miner Res. 2002 October; 17(10); 1904-12; Cotter A. and K. D.Cashman, Nutr Rev. 2003 October; 61(10): 346-51; Atkinson et at, Am JClin Nutr. 2004 February; 79(2): 326-33; Setchell K. D. and E.Lydeking-Olsen, Am J Clin Nutr 2003 September; 78(3 Suppl); 5935-609S;Clifton-Bligh et al., Menopause. 2001 July-August; 8(4): 259-65;Fitzpatrick, L. A., 2003 March 14; 44 Supl 1: S21-9). Therefore, methodsfor reducing post-menopausal bone-loss are also provided herein. In oneembodiment, such a method includes identifying a subject at risk for orsuffering from post-menopausal bone loss and administering to thesubject a therapeutically effective amount of a genistein formulationselected from any of those described herein. Alternatively, methods forpromoting bone formation are also provided. In one such embodiment, amethod for promoting bone formation, such as in the spine. Includesidentifying a subject at risk for or suffering from loss of bone massand administering to the subject a therapeutically effective amount of agenistein formulation selected from any of those described herein.

Genistein has also been suggested for use in treating cystic fibrosis.The main clinical symptoms of cystic fibrosis are chronic obstructivelung disease, which is responsible for most of the morbidity andmortality associated with cystic fibrosis, and pancreatic insufficiency.Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosistransmembrane conductance regulator (CFTR), a plasma membrane protein.CFTR functions as a chloride channel, and about 1000 mutations of thegene coding for CFTR are currently known. The most common of these knownmutations results in a deletion of a phenylalanine at position 508 ofthe CFTR protein. This mutation is referred to as Delta508 and ispresent in the majority of patients suffering from cystic fibrosis. TheDelta508 mutation results in an aberrant CFTR that is not transported tothe plasma membrane, but is instead degraded in the ubiquitin-proteasomepathway. One approach for developing a treatment for cystic fibrosis isto inhibit the breakdown of DeltaF508-CFTR by interfering with thechaperone proteins involved in the folding of CFTR. Genistein has beenshown in in-vitro systems to inhibit the breakdown of DeltaF508-CFTRthrough interference the relevant chaperone proteins. In addition, ithas been shown that it is possible to stimulate CFTR or its mutatedforms, when present in the plasma membrane, using genistein (Roomans, G.M., Am J Respir Med. 2003; 2(5): 413-31).

The genistein formulations described herein may be used in treatingcystic fibrosis. In an embodiment of such a method, a subject at riskfor or suffering from cystic fibrosis is identified and atherapeutically effective amount of a genistein formulation selectedfrom any of those described herein is administered to the subject. In aparticular embodiment, a subject at risk for or suffering from cysticfibrosis associated with DeltaF508-CFTR is identified and atherapeutically effective amount of a genistein formulation selectedfrom any of those described herein is administered to the subject. Ineach embodiment of a method for treating cystic fibrosis describedherein, the therapeutically effective amount of genistein formulationadministered to the subject is sufficient to accomplish one or more ofthe following: inhibit the breakdown of DeltaF508-CFTR; inhibit orprevent the onset of cystic fibrosis or one or more symptoms associatedwith cystic fibrosis; mitigate or reduce the severity of one or moresymptoms associated with cystic fibrosis; delay the progression ofcystic fibrosis or the worsening of one or more symptoms associated withcystic fibrosis.

Genistein appears to increase the rate at which fats are metabolized bythe body, and to decrease the rate at which they are deposited in thetissues (Goodman-Gruen, D. and D. Kritz-Silverstein, Menopause. 2003September-October; 10(5): 427-32). Moreover. In clinical studies ofhumans and animals, the consumption of genistein and daidzein resultedin loss of body fat, lower fasting insulin concentrations, lower LDL andhigher HDL cholesterol, and improved insulin responses to blood sugar.Cholesterol benefits were seen at dosages of 42 mg/day of genistein plus27 mg/day of daidzein (Bhathena, S. J. and M. T. Velasquez, Am J ClinNutr. 2002 December; 76(6): 1191-201; Urban et al., J Urol. 2001January; 165(1): 294-300). In addition to lowering LDL and raising HDL(mentioned above), genistein prevents the oxidation of LDL, a processthought to contribute to arterial plaques (Young, S. G. and S.Parthasarathy, West J Med. 1994 February; 160(2): 153-54). The genisteinformulations described herein can be used in methods for lowering LDLand/or raising HDL in subjects in need thereof. In one such embodiment,a subject at risk for or suffering from a high circulating level of LDLis identified and a therapeutically effective amount of a genisteinformulation selected from any of those described herein is administeredto the subject, wherein the therapeutically effective amount ofgenistein formulation is sufficient to lower the LDL levels or preventor delay an increase in circulating LDL levels in the subject. Inanother embodiment, a subject that could benefit from an increase incirculating levels HDL is identified and a therapeutically effectiveamount of a genistein formulation selected from any of those describedherein is administered to the subject, wherein the therapeuticallyeffective amount of genistein formulation is sufficient to increasecirculating HDL levels or prevent or delay decrease in circulating HDLlevels in the subject.

Genistein is also a radioprotective agent. For example, genistein hasbeen reported to increase hematopoiesis and survival in irradiated mice(Zhou, 2005; Land Auer, 2001, 2003 & 2005). The mechanism of action forthis radioprotective effect may potentially involve several ofgenistein's known effects including inhibition of protein tyrosinekinases (PTKs) and PTK-triggered apoptosis. Inhibition of topoisomeraseII, inhibition of phosphatidylinositol turnover and the second messengersystem, both agonist and antagonist estrogenic effects, reduction ofstress gene expression through inactivation of Y/CCA-AT binding factor.Increased antioxidant activity, apoptosis, cell cycle arrest anddifferentiation, improved immune defenses and/or increased AKT kinaselevels. The beneficial effects of genistein may also be due. In part, toits antioxidant properties, reducing free radicals and stabilizing thecell membrane structure. Further, genistein may also have a role inprotecting stem cells and/or stimulating proliferation.

Genistein administered prior to, during, and/or after exposure toradiation, may be used to eliminate or reduce the severity ofdeleterious cellular effects caused by exposure to ionizing radiationresulting from, for example, from a nuclear explosion, a spill ofradioactive material, close proximity to radioactive material, cancerradiation therapy, diagnostic tests that utilize radiation, and thelike. Genistein can be used for the treatment and prevention of AcuteRadiation Syndrome (ARS) (sometimes known as radiation toxicity orradiation sickness). ARS is an acute illness caused by irradiation of asubstantial portion of the body by a high dose of penetrating radiation(Le., greater than 0.7 Gray (Gy) or 70 rads, with mild symptoms possibleat doses as low as 0.3 Gy or 30 rads) over a very short period of time(usually a matter of minutes). It is thought that the major cause of ARSis depletion of immature parenchymal stem cells in specific tissues.

Methods for treating radiation exposure are, therefore provided herein.In each embodiment, a subject at risk of or that has suffered fromexposure to radiation is identified and a therapeutically effectiveamount of a genistein formulation selected from any of those describedherein is administered to the subject. In specific embodiments, themethod of treating radiation exposure is a method for preventing ARS,wherein a subject at risk of ARS is identified and a therapeuticallyeffective amount of a genistein formulation as described herein isadministered to the subject before the subject is exposed to radiation.In other embodiments, the method of treating radiation exposure is amethod for treating ARS, wherein a subject suffering from ARS isidentified and a therapeutically effective amount of a genisteinformulation as described herein is administered to the subject after thesubject has suffered exposure to radiation. In yet other embodiments, asubject at risk of radiation exposure is identified, a therapeuticallyeffective amount of a genistein formulation as described herein isadministered to the subject prior to exposure to radiation, and. In theevent the subject suffers from radiation exposure, administration oftherapeutically effective amounts of genistein is continued after theradiation exposure occurs,

In additional embodiments, subjects at risk for or having suffered froma radiation exposure resulting from an event selected from cancerradiation therapy or a diagnostic test utilizing radiation areidentified, and the subjects are administered a therapeuticallyeffective amount of the genistein formulation. In one such embodiment,the genistein formulation is administered to the subject prior toradiation exposure in order to prevent or reduce the severity of thedeleterious effects of such exposure. In another such embodiment, thegenistein formulation is administered to the subject after radiationexposure in order to mitigate, reverse or reduce the severity of thedeleterious effects of such exposure. In still another embodiment, themethods of treating radiation exposure resulting from an event selectedfrom cancer radiation therapy or a diagnostic test utilizing radiationin a subject may include administering a genistein formulation asdescribed herein both before and after radiation exposure.

In each of the embodiments of the methods described herein, thetherapeutically effective amount of genistein formulation may beadministered orally or parenterally. In specific embodiments, where thegenistein formulation is administered parenterally, it may beadministered, for example, via intravenous injection or infusion,subcutaneous injection. Intravascular injection, or intramuscularinjection. Where the formulation is administered orally, the formulationmay be prepared in any manner suitable for oral administration, such asis described herein. The dose and dosing regimen most appropriate for agiven embodiment of the therapeutic methods described herein may dependupon, for example, the subject being treated, the nature of the diseaseor disorder, as well as the severity of any symptoms suffered. Usingformulations prepared as described herein, one of skill in the art willbe able to identify the appropriate dose and dosing regimen useful forachieving therapeutic efficacy in each of the methods described herein.The genistein formulations described herein may be administered, forexample, as a single dose, a regular daily dose, a two-times daily dose,a three-times daily dose, or according to another desired dosingschedule.

The total daily dose of genistein delivered using a formulation ormethod described herein may depend on the desired condition to betreated or the desired therapuetic effect. In specific embodiments, atherapeutically effective amount of a genistein formulation according tothe present description may be an amount sufficient to deliver a dose ofgenistein ranging from about 50 mg/day to about 10,000 mg/day. Incertain such embodiments, the amount of genistein formulationadministered to the subject is sufficient to deliver a dose of genisteinselected from about 50 mg/day to about 9,000 mg/ day, about 50 mg/day toabout 8,000 mg/ day, about 50 mg/day to about 2,000 mg/day, about 100mg/day to about 9,000 mg/day, about 100 mg/day to about 5,000 mg/day,about 100 mg/day to about 4,000 mg/ day, and about 100 mg/day to about2,000 mg/day.

EXAMPLES Example 1 Solubility of Genistein

Calculated pKa's for genistein range from 7-9, with the predictedsolubility increasing above pH 7 in accordance with the lowest pKa. Thecalculated properties were used to design the appropriate pH range forthe pH-solubility profile of genistein in several acceptable cosolvents,which was established to be pH 6-9. Solubility of genistein wasincreased at higher pH, however degradation was observed at pH 9, Table1 shows the solubility results of genistein in selected pharmaceuticallyacceptable cosolvents.

TABLE 1 Solubility at 25° C. Vehicle (mg/mL) Propylene Glycol 6.2Polyethylene Glycol 300 110.5 (PEG300) Polyethylene Glycol 400 115.1(PEG400) Ethanol 25.0 Dimethyl acetamide (DMA) 141.3 N-methylpyrrolidone (NMP) 238 Citrate buffer pH 6 Not detected Phosphate bufferpH 7 Not detected TRIS buffer pH 8 Not detected Borate buffer pH 9, 3days Not detected Borate buffer pH 9, 7 days 0.043 Borate buffer pH 9,14 days 0.005

The solubility of genistein in water is not detectable at pH 6-7, whichimplies the solubility is less than 0.02 mg/mL or ˜0.00002 g/g H₂O(lowest concentration). Based on the pH-solubility and solubility incosolvents, it was determined that PEG400 would be the cosoivent toachieve highest solubility. Since parenteral formulations preferablyhave a maximum of 50% organic component, the addition of Ethanol (EtOH),Nmethylpyrrolidone (NMP) and a surfactant were considered, since thesewould be expected to enhance absorption from the injection site. Ethanolhas the added benefit of reducing viscosity. Polysorbate 80 (Tween 80)was considered as a surfactant due to its use in approved parenteraldosage forms at levels as high as 12% (FDA Inactive Ingredients Guide),although a more common range is 0.1-1%. Solubility was further evaluatedin two aqueous/organic mixtures with concentrations acceptable for aparenteral dosage form. Additionally, cyclodextrin formulations wereevaluated. Solubility testing results for genistein are given in Table2.

TABLE 2 Solubility of GENISTEIN at 25° C. Vehicle (mg/mL) 10%Polysorbate 80/40% PEG400/50% 25 mM 11 Phosphate Buffered Saline (pH 7)10% Polysorbate 80/10% EtOH/40% PEG400/40% 12 25 mM Phosphate BufferedSaline (pH 7) 10% Polysorbate 80/10% NMP/40% PEG400/40% 25 mM 19Phosphate Buffered Saline (pH 7) 30% Hydroxypropyl-β-cyclodextrin 6 30%Sulfobutylether-β-cyclodextrin 8

Example 2 Nanoparticulate Genistein Composition

None of the previously evaluated genistein formulations achieved thedesired level of drug loading. In an effort to achieve higher drugloading, a sterile injectable suspension was prepared according to thepresent description. The formulation included nanoparticulate genisteinthat had been nanomilled with a vehicle solution of 5% Povidone K17(w/w), 0.2% Polysorbate 80 (w/w). In 50 mM phosphate buffered saline (61mM sodium chloride). The quantitative composition of the formulation islisted in Table 3.

TABLE 3 Component Amount Amount per 1 L Genistein 300 mg/mL 300 gPolysorbate 80  2 mg/mL  2 g Povidone K17  40 mg/mL  50 g 50 mM Sodium0.948 mg/mL   948 g Phosphate/61 mM Sodium Chloride

The function of each component and excipient listed in Table 1 is asfollows: 1) Polysorbate 80 is included as a surfactant to enable wettingand aid in preventing agglomeration of suspended genistein drugsubstance, 2) Povidone K17 is included as a viscosity enhancer to aid instabilizing the genistein drug substance suspension, and 3) SodiumPhosphate Buffer, Sodium Chloride is included as the diluent and toachieve physiological osmolality and maintain pH for parenteraladministration of the composition.

The composition of 50 mM Sodium Phosphate Buffer/61 mM Sodium Chloridesolution is as shown in Table 4.

TABLE 4 Component Amount per 1 L NaH₂PO₄•H₂O  6.9 g NaCl 3.56 g NaOH (pHadjust) HCl (pH adjust)

Example 3 Second Nanoparticulate Genistein Composition

A second nanoparticulate genistein formulation as described herein wasprepared. The nanomilled genistein was achieved using wet bead milling,utilizing an agitator bead mill in a horizontal grinding container forcontinuous dispersion and fine wet grinding in a closed system. ADYNO®-Mill Type Multi Lab agitator bead mill was used to prepare thenanoparticulate genistein, wherein the necessary energy for dispersionand grinding was transmitted to the grinding beads via the agitatordiscs mounted on the agitator shaft. Material was continuously fed intothe mill via a product pump. The gap setting of the dynamic gapseparator, the diameter of the beads, and length of the milling periodwere used to determine the particle size distribution. The product wasfed continuously through the mill until a defined particle sizedistribution was reached. Although the DYNO®-Mill Type Multi Labagitator bead mill was utilized in this work, other high energy, wetbead milling process equipment may be utilized.

Two formulations were tested that incorporated either Polysorbate 80 orPoloxamer 188 as a wetting agent to maintain a stable particle sizedistribution. Povidone (Polyvinyl pyrrolidone (PVP)) K17 was used at the5% level in the formulations as a viscosity enhancer as well as astabilizer against particle agglomeration. The quantitative compositionof the formulation is given in Table 5.

TABLE 5 Reference to Quality Component Standard Amount (mg/mL) GenisteinSP-001 300 Polysorbate 80 USP 2 Povidone K17 USP 50 Sterile Water forInjection USP QS to 1 mL

The formulation may also include the replacement of the sterile waterwith injection with a phosphate-buffered saline for pH control andosmolality (e.g., as provided in the formulation described in Example2).

The formulations exhibited an excellent, reproducible and stableparticle size distribution profile, with d(0.5) of less than 0.2 μm.Optical microscopy confirmed a uniform particle size in the suspension,Powder X-Ray diffraction (XRD) was performed to examine physical,crystalline changes to the genistein material as a result of the millingprocess or as a result of a formulation incompatibility. Analysesperformed indicate that there was no change in crystal form post-millingfor genistein drug substance and milled suspensions containing 0.2%(w/w) polysorbate 80 with 5% (w/w) povidone K17, and 0.2% (w/w)poloxamer 188 with 5% (w/w) povidone K17.

The nanomilled genistein suspension comprised of nanoparticulategenistein (300 mg/mL) containing 0.2% (w/w) Polysorbate 80 with 5% (w/w)Povidone K17 was placed on stability at 5° C., 30° C. at 65% RH, and 40°C. at 75% RH. The suspensions were stored in 5 mL serum vials and 20 mmPTFE-faced butyl rubber stoppers. No impurities were observed after 7months and there was no significant change in the particle sizedistribution.

Example 4 In-Vivo Comparison of Genistein Suspension Formulation withGenistein Solution Formulation

This experiment evaluated a nanoparticulate formulation of genisteinaccording to the present description and compared it to administrationof genistein in a PEG 400 solution formulation. The genistein suspensionformulation included nanomilled genistein in 50 nM phosphate bufferedsaline with 0.2% (w/w) Polysorbate 80 and 5% (w/w) PVP K17. Thesuspension formulaiton exhibited a pH of 6.96, and the nanoparticulategenistein incorporated into the suspension formulation exhibited a D(0.50) of 0.126 μm and a D (0.90) of 0.253 μm. The formulations wereadministered via subcutaneous injection(“SC”) 24 hr prior toirradiation. A separate vehicle and genistein group was included foreach formulation. The study was conducted at two radiation doses,either, 8.75 Gy or 9.0 Gy.

Male CD2F1 mice were exposed to bilateral whole-body irradiation at adose of 8.75 Gy or 9.0 Gy at 0.6 Gy/min. Thirty-day survival was theendpoint for this study. The different experimental groups evaluated inthis study are detailed in Table 6.

TABLE 6 Experimental Groups: 30 Day Survival Group Route Time of SC Gy N(%) At 8.75 Gy: 1.) Vehicle, PEG-400 SC −24 Hr pre-rad 8.75 16 38% 2.)Genistein (PEG-400) SC −24 Hr pre-rad 8.75 16  81%* 3.) Vehicle (Nano))SC −24 Hr pre-rad 8.75 16 25% 4.) Genistein (Nano) SC −24 Hr pre-rad8.75 16 100%* At 9.0 Gy: 1.) Vehicle, PEG-400 SC −24 Hr pre-rad 9.0 1638% 2.) Genistein (PEG-400) SC −24 Hr pre-rad 9.0 16  81%* 3.) Vehicle(Nano) SC −24 Hr pre-rad 9.0 16 19% 4.) Genistein (Nano) SC −24 Hrpre-rad 9.0 16  88%*

As shown in Table 6 and FIG. 1, the thirty-day survival rates in groupsreceiving solution formulation of genistein (Genistein (PEG-400)) andsuspension formulation of genistein (Genistein (NANO)) at 8.75 Gy were81% and 100%, respectively. Survival rates of the control groups(Vehicle (PEG-400) and Vehicle (Nano)) were 38% and 25%, respectively.At 9.0 Gy, 30-day survival rates of the Genistein (PEG-400) group andthe Genistein (NANO) group were 81% end 88% respectively. The survivalrates of the control groups (Vehicle (PEG-400) and Vehicle (Nano)) were38% and 19%, respectively. Every group that received genistein 24 hrpre-irradiation were significantly (p<0.05) different from theirrespective control group.

Example 5 In-Vivo Comparison Genistein Suspension FormulationAdministered Parenteral and Genistein Suspension Formulation andGenistein Solution Formulation Given Orally

This experiment evaluated the effect of a genistein nanoparticulatesuspension formulation (Gerstein-IS) prepared as described in Example 4given via intramuscular injection (“IM”) compared to the effect of a PEG400 solution formulation and the Genistein IS suspension formulationgiven orally. The different formulations were administered twice dailyfor 6 days prior to irradiation. A positive control was also includedwhich was Genistein-IS administered IM 24 hours prior to irradiation. Aseparate vehicle and genistein group was included for each group. Thestudy was conducted at one radiation dose, 9.25 Gy. Male CD2F1 mice wereexposed to bilateral whole-body irradiation at a dose of 9.25 Gy at 0.6Gy/min Thirty-day survival was the endpoint for this study.

TABLE 7 Experimental Groups: 30 Day Survival Group Route Time of SC Gy N(%) 1) Vehicle, IS IM −24 Hr pre-rad 9.25 20 10%  2) Genistein IS IM −24Hr pre-rad 9.25 20 85%* 3) Vehicle, IS PO BID for 6 days pre-rad 9.25 2015%  4) Genistein IS PO BID for 6 days pre-rad 9.25 20 85%* 5) Vehicle,PEG PO BID for 6 days pre-rad 9.25 20 0% 400 6) Genistein/PEG PO BID for6 days pre-rad 9.25 20 80%* 400 p < 0.05 two-tailed Fisher Exact Test(vehicle vs. genistein) BID = twice daily dosing IS = InjectableSuspension

As shown in Table 7, the thirty-day survival rates of orallyadministered Genistein/PEG 400 and Genistein-IS at 9.25 Gy were 80% and85%, respectively. Survival rates of the control groups (vehicle onlyadministration) were 0% and 15%, respectively. The positive controlgroup, Genistein IS administered IM 24 hours prior to irradiation, had asurvival percentage of 85% vs. 10% for the vehicle.

Every group that received genistein either IM or orally weresignificantly (p<0.05) different from their respective negative controlgroup. There was not, however, a significant difference in survivalbetween genistein/PEG-400, and the Genistein IS formulation,

Example Radio Protection Time Course Study with Vehicle InjectionSuspension and Genistein Nano Particulate Injection SuspensionSubcutaneously Administered 24 Hr, 18 Hr, 12 Hr, or 6 Hr Before 9.0 Gy⁶⁰Co Radiation

Previous experiments showed statistically significant radioprotectiveresults when a nanoparticulate genistein injectable suspension preparedaccording to the present description (Genistein-IS) was administered 24hr before irradiation in a saline based vehicle. This time course studywas performed to determine whether there was a time-dependent effect onradioprotective efficacy with SC administered Genistein-IS. The timedependent effects of Genistein-IS were compared to a placebo formulation(Vehicle-IS). The Genistein-IS formulation was prepared as described inExample 4.

Male CD2F1 mice were used in this experiment. All groups received asingle 200 mg/kg SC administration at 24 hr, 18 hr, 12 hr, or 6 hrbefore irradiation. SC injections were administered in the nape of theneck using a 25 G needle in an injection volume of 0.1 ml via a 1 mltuberculin syringe. All mice were exposed to bilateral whole-bodyirradiation at a dose of 9.0 Gy at 0.6 Gy/min. Thirty-day survival wasthe endpoint for this study.

TABLE 8 Experimental Groups: 30-Day Survival Group Route Time of Dose GyN (%) 1.) Vehicle-IS SC 24 Hr pre-rad 9.0 16 44% 2.) Genistein-IS SC 24Hr pre-rad 9.0 16  88%* 3.) Vehicle-IS SC 18 Hr pre-rad 9.0 16 13% 4.)Genistein-IS SC 18 Hr pre-rad 9.0 16  69%* 5.) Vehicle-IS SC 12 Hrpre-rad 9.0 16 44% 6.) Genistein-IS SC 12 Hr pre-rad 9.0 16 81% 7.)Vehicle-IS SC  6 Hr pre-rad 9.0 16 38% 8.) Genistein-IS SC  6 Hr pre-rad9.0 16 63% *p < 0.05 two-tailed Fisher Exact Test (vehicle vs.genistein)

The results shown in Table 8 and FIG. 2 demonstrate that a single SCadministration of Genistein-IS administered at 24, 18, 12 , or 6 hrpre-irradiation resulted in 30-day survival rates of 88%, 69%, 81%, and63%, respectively. The survival rates for the Vehicle-IS groups at thecorresponding time points were 44%, 13%, 44%, and 38%, respectively.Genistein-IS resulted in significant radioprotection when injectedeither 24 or 18 hr before irradiation (p<0.05).

Example 7 Effect of Subcutaneous vs. Intramuscular Injection ofNanoparticulate Genistein Formulation on Radioprotective Efficacy WhenAdministered 24 hr Before 9.25 Gy⁶⁰Co Radiation

The purpose of this experiment was to compare the radioprotectiveefficacy of a nanoparticulate genistein suspension formulation preparedas described herein (Genistein-IS) delivered to provide a genistein doseof 200 mg/kg when administered SC or IM. The Genistien-IS formulationwas prepared as described in Example 4. Male CD2F1 mice were used inthis experiment. Groups were given a single SC or IM injection of aninjectable placebo suspension (Vehicle-IS) or Genistein-IS (200 mg/kg)24 hr before irradiation. Also included in this experiment were groupsthat received a solution formulation of genistein in PEG 400 (Genistein)delivered to provide a genistein dose of 200 mg/kg or placebo PEG 400formulation (PEG 400) administered SC 24 hr before irradiation.

All vehicle and Genistein-IS groups received a single 200 mg/kg SC or IMinjection at 24 hr before irradiation. SC injections were administeredin the nape of the neck using a 25 G needle in an injection volume of0.1 ml via a 1 ml tuberculin syringe. Mice were administered Vehicle-ISor Genistein-IS by IM injection into the quadriceps muscle using a 25 Gneedle attached to a Hamilton syringe. The injection volume was 50 ∥l.

Mice were exposed to bilateral whole-body irradiation at a dose of 9.25Gy at 0.6 Gy/min. Thirty-day survival was the endpoint for this study.

TABLE 9 Experimental Groups: 30-Day Survival Group Route Time of Dose GyN (%) 1.) PEG 400 SC 24 Hr pre-rad 9.25 20 15%  2.) Genistein SC 24 Hrpre-rad 9.25 20 75%* 3.) Vehicle IS SC 24 Hr pre-rad 9.25 20 30%  4.)Genistein IS SC 24 Hr pre-rad 9.25 20 85%* 5.) Vehicle IS IM 24 Hrpre-rad 9.25 20 15%  6.) Genistein IS IM 24 Hr pre-rad 9.25 20 75%* *p <0.05 two-tailed Fisher Exact Test (vehicle vs. genistein)

Survival rates for vehicle-PEG 400 and genistein PEG 400 administered SCwere 15% and 75%, respectively. This resulted in statisticallysignificant radioprotection by genistein over PEG 400 vehicle (p<0.05)(shown in Table 9 and FIG. 3).

When Vehicle-IS or Genistein-IS was administered SC, 30-day survivalrates were 30% and 85%, respectively. For IM administration, survivalrates for Vehicle-IS and Genistein-IS, were 15% and 75%, respectively.For both SC and IM routes, genistein provided significant protectionover vehicle (p<0.05) (shown in Table 9 and FIG. 3).

These results demonstrate that nanoparticulate genistein formulationsprepared according to the present description provide similar levels ofradioprotection when administered by either the SC or IM route.

Example 8 Pharmacokinetics in Mice Following Intravenous orintramuscular Injection of ¹⁴C-Genistein

As shown in the results of Table 10, male CD1 mice were administered asingle IM dose (Group 2, nominal 200 mg/kg) or a single IV bolus dose(Group 3, nominal 50 mg/kg) of ¹⁴C-Genistein. The genistein formulationused was a supsension formulation that includes genistein suspended insterile water with 0.2% (w/w) Polysorbate 80 and 5% (w/w) PVP K17. Thegenistein material used in the suspension formulation exhibited a D(0.50) of 0.136 μm and a D(0.90) of 0.310 μm. Following dosing, thecontent and concentration of radioactivity in blood, plasma, excreta andcarcass, and the non-compartmental pharmacokinetics of totalradioactivity in whole blood and plasma were determined. Following doseadministration, the extent and severity of any clinical signs wasassessed. The dose level of 200 mg/kg (IM) and 50 mg/kg (IV) were welltolerated and therefore chosen for the main study.

The concentration of radioactivity in both dose formulations wasmeasured pre and post dose by liquid scintillation spectroscopy and wassimilar on both occasions. The radiochemical stability of the testarticle in both dose formulations was assessed prior to and followingthe administration of the dose. The mean radiochemical stability of thetest article in the dose formulation samples from the intramuscular doseformulation (Group 2) were 98.5% and 98.2%, respectively. The meanradiochemical stability values for pre and post dose samples from theintravenous dose formulation (Group 3) were 98.3% and 98.5%,respectively. Therefore, the ¹⁴C-Genistein in both formulations wasconsidered to have been radiochemically stable throughout the dosingperiod. No treatment-related clinical signs were observed in any of themain study male mice following a single IM dose of ¹⁴C-Genistein (200mg/kg) or a single IV dose of ¹⁴C-Genistein (50 mg/kg).

Whole-blood samples were collected and plasma was obtained bycentrifugation. The concentration of radioactivity in whole-blood andplasma was measured by liquid scintillation spectroscopy.Pharmacokinetic parameters were calculated from the compositeconcentration vs. time profiles and are presented in Table 10.

TABLE 10 Pharma- cokinetic Group 2 (IM) Group 3 (IV) Parameter UnitsBlood Plasma Blood Plasma t_(max) h 0.50 0.50 0 0 C_(max) μg 33.3 63.160.1 108 eq/mL t_(last) h 168 168 168 168 AUC_(0-tlast) μg 222 375 99.7116 eq · h/ mL R² — 0.977 0.943 0.850 0.999 k_(e1) h⁻¹ 0.0242 0.01450.0118 0.0307 t_(1/2) h 28.7 47.8 58.5 22.6 AUC_(0-inf) μg eq · 223 387112 116 h/mL Extra- % 0.663 3.18 11.4 0.191 polation V_(d) mL/kg NA NA37554 14042 CL mL/h/ NA NA 445 431 kg Bio- % 49.7 83.5 NA NAavailibility

For Group 2, the highest mean concentration of radioactivity in plasmaand whole blood was observed at 30 minutes post dose (first time pointanalyzed), suggesting a rapid absorption from the IM dose. Blood toplasma ratios of less than 1 suggested that dose-related material wasnot particularly associated with the blood cells at any time post dose.Exposure of plasma to dose-related material was greater than that ofwhole blood, as measured by AUC_(0-Inf), and the rate of clearance wasslower (as measured by t_(1/2)). The systemic exposure (AUC_(0-Inf))following the IM administration was relatively good with an estimationof relative bioavailability of total radioactivity of 49.7% and 83.5%for blood and plasma, respectively.

For Group 3, the highest mean concentration of radiolabelled material inplasma and whole blood was observed at 30 minutes post dose (the firsttime point analyzed). For the early time points (0 to 24 hours),concentrations in plasma were always higher than those in blood, asreflected by blood-to-plasma ratios of less than 1. This indicated thatdose-related material was not particularly associated with the bloodcells at these time points. After 24 hours, the concentrations ofradioactivity in blood were always higher than those in plasmasuggesting that the dose-related material was associated with the bloodcells. Exposure of plasma to dose-related material was similar to wholeblood, as measured by AUC_(0-Inf), but the rate of clearance was fasteras measured by t_(1/2).

The major route of excretion following an IM dose or an IV bolus dosewas via urine, with a smaller percentage recovered in feces. Therecoveries in excreta following the intramuscular and intravenous doseswere very similar, at approximately 52.5% to 54.0% for urine and atapproximately 31.3% to 35.5% for feces. For both dose routes, excretionwas relatively rapid with the majority of the dose administered excretedwithin 24 hours. The proportion of the administered radiolabelledmaterial recovered in feces suggested that biliary excretion ofdose-related material had occurred following both dose routes. Excretionrecovery was approximate 92% and 93% for Groups 2 and 3, respectively.Indicating that excretion was essentially complete by 168 hours postdose. A small percentage of the administered radiolabelled material, forboth routes, was found in the remaining carcass. Thus the overall meanmass balance of radioactivity was good, at approximately 93% to 94% ofthe administered dose for both Groups 2 and 3 animals.

In conclusion, male mice were administered an IM dose (200 mg/kg) or anIV bolus dose (50 mg/kg) of ¹⁴C-Genistein. Concentrations ofradioactivity in whole blood, plasma, excreta and carcass weredetermined. The highest radioactivity concentrations were observed at 30minutes post intramuscular or intravenous bolus dose. Indicating rapidabsorption from the intramuscular dose. The bioavailability ofdose-related material following the intramuscular dose was good, atgreater than 49%. Radioactivity was excreted rapidly and urine was themajor route of excretion for both dose routes. The high level ofradioactivity recovered in feces following the intramuscular orintravenous bolus dose suggested that biliary excretion had occurred.The total recovery of dose-related material following both dose routeswas essentially complete by 168 hours post dose.

Example 9 Pharmacokinetics in Beagle Dogs Following Intravenous orIntramuscular Injection of ¹⁴C-Genistein

Male Beagle dogs were administered a single IV bolus dose (Group 1,nominal 20 mg/kg) or a single IM dose (Group 2, nominal 20 mg/kg) of₁₄C-Genistein (results shown in Table 11). The Genistein suspensionformulation used was prepared as described in Example 8. Followingdosing, the content and concentration of radioactivity in blood, plasmaand excreta, and the non-compartmental pharmacokinetics of totalradioactivity in whole blood and plasma were determined. Theconcentration of radioactivity in both dose formulations was measuredpre and post dose by liquid scintillation spectroscopy and was similaron both occasions. The radiochemical stability of the test article inboth dose formulations was assessed prior to and following theadministration of the dose. The mean stability values for pre and postdose samples from the intravenous dose formulation (Group 1) were 100%and 99.6%, respectively. The mean stability values for pre and post dosesamples from the intramuscular dose formulation (Group 2) were 99.2% and98.9%, respectively. Therefore, the ¹⁴C-Genistein in both formulationswas considered to have been radiochemically stable throughout the dosingperiod.

Whole-blood samples were collected and plasma was obtained bycentrifugation. The concentration of radioactivity in whole-blood andplasma was measured by liquid scintillation spectroscopy.Pharmacokinetic parameters were calculated from the concentration vs.time profiles and are presented in Table 11.

TABLE 11 Pharma- cokinetic Group 1 (IV) Group 2 (IM) Parameter UnitsBlood Plasma Blood Plasma t_(max) h 0 0 2 2 C_(max) μg 40.4 94.4 5.3910.9 eq/mL t_(last) h 120 120 168 168 AUC_(0-tlast) μg eq · 117 201 108193 h/mL k_(e1) h⁻¹ 0.0130 0.0138 ^(a) 0.0092 t_(1/2) h 55.7 227 ^(a)75.2 AUC_(0-inf) μg eq · 122 227 ^(a) 228 h/mL Extrapolation % 16.8 11.3^(a) 15.1 V_(z) mL/kg 13168 6511 NA NA CL mL/h/ 163 88.7 NA NA kgBioavailibility % NA NA 92.9 96.1 ^(a)extrapolation to AUC_(0-inf)greater than 20%, therefore not reported. NA not applicable

For Group 1 (IV bolus dose), the highest mean concentration ofradiolabelled material in plasma and whole blood was observed at 15minutes post dose (the first time point analyzed). Concentrations inplasma were always higher than those in blood, as reflected by blood toplasma ratios of less than 1. This indicated that dose-related materialwas not particularly associated with the blood cells at these timepoints. Exposure of plasma to dose-related material was greater thanthat of whole blood, as measured by AUC_(0-inf), but the rate ofclearance was similar as measured by t_(1/2).

For Group 2 (IM dose), the highest mean concentration of radioactivityin plasma and whole blood was observed at 2 hours post dose, suggestinga relatively rapid absorption from the intramuscular dose. Blood toplasma ratios of less than 1 suggested that dose-related material wasnot particularly associated with the blood cells at any time post dose.Exposure of plasma to dose-related material was greater than that ofwhole blood, as measured by AUC_(0-tlast). The rate of clearance (asmeasured by t_(1/2)) was generally slower than the one observedfollowing the IV bolus dose. However, the systemic exposure(AUC_(0-tlast)) following the IM administration was good with anestimation of relative bioavailability of total radioactivity of 92.9%and 96.1% for blood and plasma, respectively.

The major route of excretion following an intravenous bolus dose or anIM dose was via feces, with a smaller percentage recovered in urine. Therecoveries in excreta following the IV bolus and IM doses were verysimilar, at approximately 48.7 to 51.9% for feces and at approximately32.0 to 33.6% for urine. For both dose routes, excretion was relativelyrapid with the majority of the dose administered excreted within 48hours. The proportion of the administered radiolabelled materialrecovered in feces suggested that biliary excretion of dose-relatedmaterial had occurred following both dose routes. Excretion recovery by168 hours post dose was approximately 87.9% and 85.8% for Groups 1 and2, respectively. Thus, the overall mean excretion mass balance ofradioactivity for both groups was good, at approximately 86-88% of theadministered dose.

In conclusion, male dogs were administered an IV bolus dose (20 mg/kg)or an IM dose (20 mg/kg) of ¹⁴C-Genistein. Concentrations ofradioactivity in whole blood, plasma and excreta were determined.Clinical signs were observed in both groups and were considered to bedose-related. The highest radioactivity concentrations in blood andplasma were observed at 15 minutes (intravenous dose) or 2 hours(intramuscular dose) post dose. Indicating relatively rapid absorptionfrom the IM dose. The bioavailability of dose-related material followingthe IM dose was good, at greater than 92%. Test article-related materialwas excreted rapidly and feces was the major route of excretion for bothdose routes. The high level of radioactivity recovered in fecesfollowing the IV bolus or intramuscular dose suggested that biliaryexcretion had occurred. The excretion mass balance for both dose routeswas considered good at greater than 85%.

Example 10 Oral Pharmacokinetic Comparison Study

An oral bioavailability comparison of genistein solution formulation inPEG 400 vs. a genistein nanosuspension prepare according to the presentdescription was carried out. The genistein suspension formulation wasprepared as described in Example 4. Considering the limited oralbioavailability of genistein in earlier preclinical and clinical work,this experiment was designed to compare a previously used genisteinsolution formulation prepared with PEG 400 as the vehicle with agenistein nanosuspension formulation prepared as described herein.

Ten groups of seven mice were prepared at each time point (70 mice) foreach of the two formulations (total mice=140). A single dose of 400mg/kg genistein was given by oral gavage and then blood was collected at10 subsequent time points. Time points for blood collection were thefollowing: 0, 0.5, 1, 2 , 3 , 4 , 6, 8 , 10 and 12 hours postadministration.

The oral bioavailability with the nanoparticulate suspension wasstatistically significantly greater than that achieved in the PEG 400solution formulation. Both free and total genistein levels weredetermined in each of the groups for every time point The meanconcentration of seven mice was determined for each time point andreported. At the time 0 time point, the free and total genisteinconcentration was below the limit of quantitation and not reported. Forthe mice receiving the nanoparticulate suspension, the free genisteinconcentrations were significantly greater at 1, 2, 4, 8, 10 and 12 hourswhen compared to the concentration achieved in the mice receiving thePEG 400 solution formulation. The total genistein concentrations werealso significantly greater with the nanoparticulate suspension at 0.5,1, 2, 4, 8. 10 and 12 hours when compared to the PEG 400 solutionformulation. As noted in Table 12 and Table 13, and as shown in FIG. 4and FIG. 5, the absorption and excretion curve for the nanoparticulatesuspension is also much more predictable and less erratic than thatachieved by the PEG 400 formulation for both the free and totalgenistein concentrations. Free genistein determinations after a singleoral dose of 400 mg/kg for two different genistein formulations in mice.

TABLE 12 Mean Free Genistein Concentrations at Each Sampling Time pointFree Free Time PEG 400 Nanosuspension point (ng/ml) (ng/ml) DifferencePercent p value 0.5 hr   1137.9 1598.6 460.7 40.5 0.062 1 hr 514.0 999.7485.7 94.5 0.006 2 hr 255.1 746.4 491.3 192.6 0.000 3 hr 403.1 634.6231.4 57.4 0.079 4 hr 207.6 484.3 276.7 133.2 0.002 6 hr 361.9 313.6−48.4 −13.4 0.791 8 hr 436.3 172.3 −264.0 −60.5 0.023 10 hr  239.0 59.0−180.0 −75.3 0.000 12 hr  113.7 28.3 −85.4 −75.1 0.034

TABLE 13 Mean Total Genistein Concentrations at Each Sampling Time pointTotal Total Time PEG 400 Nanosuspension point (ng/ml) (ng/ml) DifferencePercent p value 0.5 hr   15640.0 32114.3 16474.3 105.3 0.000 1 hr 7711.420974.3 13262.9 172.0 0.002 2 hr 4151.4 12050.0 7898.6 190.3 0.033 3 hr6251.4 9112.9 2861.5 45.8 0.136 4 hr 2970.1 7692.9 4722.8 159.0 0.003 6hr 6215.1 4004.3 −2210.8 −35.6 0.257 8 hr 5558.6 1982.9 −3575.7 −64.30.006 10 hr  3480.0 755.4 −2724.6 −78.3 0.001 12 hr  2205.0 380.3−1824.7 −82.8 0.002

Example 11 Oral Bioavailability Comparison of Non-nanoparticulateGenistein Suspension Formulation and a Nanoparticulate GenisteinFormulation

In a previous experiment we were able to demonstrate improved oralbioavailability with orally administered Genistein-IS suspensionformulation in comparison with a formulation of genistein dissolved inPEG-400. This experiment compared the oral bioavailablity of an aqeousgenistein suspension formulation that included non-nanoparticulategenistein material with that provided by the Genistein-IS formulationprepared as described in Example 4. The non-nanoparticulate genistensuspension formulation was the same as the Genistein-IS formulation,except that the genestein material exhibited a volume average particlesize of 8 μm. Instead of the volume average particle size of 0.13 μmexhibited by the Genistein-IS suspension formulation.

Ten groups of seven mice were prepared at each timepoint (70 mice) foreach of the two formulations (total mice=140). A single dose of 400mg/kg was given by oral gavage and then blood was collected at 10subsequent timepoints. Time points for blood collection were thefollowing: 0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours postadministration.

Both free and total genistein levels were determined in each of thegroups for every timepoint. The mean concentration of seven mice wasdetermined for each timepoint and reported. At the time 0 timepoint, thefree and total genistein concentration was below the limit ofquantitation and not reported. For the Genistein-IS suspensionformulation mice, the free genistein concentrations were significantlygreater at 0.5, 1 & 2 hours when compared to those achieved by thenon-nanoparticulate genistein formulation (See, Table 14). The totalgenistein concentrations were also significantly greater with the withthe Genistein-IS suspension formulation at 0.5, 1 and 2 hours whencompared to those achieved by the non-nanoparticulate genisteinformulation (See, Table 15). As noted in FIG. 6 and FIG. 7, theabsorption and excretion curve for the Genistein-IS suspensionformulation is also much more predictable and less erratic than thatachieved by the non-nanoparticulate genistein formulation for both thefree and total genistein concentrations.

TABLE 14 Mean Free Genistein Concentrations at Each Sampling TimepointFree Conc. Free Conc. 8 Micron Nanosuspension p Timepoint (ng/ml)(ng/ml) Difference Percent value 0.5 hr   630.7 1494.0 863.3 136.9 0.0001 hr 512.1 853.4 341.3 66.6 0.003 2 hr 314.4 652.6 338.1 107.5 0.003 4hr 214.3 367.4 153.1 71.5 0.055 6 hr 136.3 168.0 31.7 23.2 0.460 8 hr140.1 45.8 −94.3 −67.3 0.006 10 hr  50.3 21.6 −28.6 −57.0 0.284 12 hr 47.3 23.9 −23.3 −49.4 0.195 24 hr  0.0 0.0 0.0 — —

TABLE 15 Mean Total Genistein Concentrations at Each Sampling TimepointTotal Conc. Total Conc. 8 Micron Nanosuspension p Timepoint (ng/ml)(ng/ml) Difference Percent value 0.5 hr   9561.4 32385.7 22824.3 238.70.000 1 hr 9762.9 25842.9 16080.0 164.7 0.000 2 hr 5247.1 15357.110110.0 192.7 0.007 4 hr 3230.0 5938.6 2708.6 83.9 0.055 6 hr 2230.42423.1 192.7 8.6 0.802 8 hr 1902.9 1007.1 −895.7 −47.1 0.070 10 hr 1002.2 426.4 −575.8 −57.5 0.253 12 hr  876.4 338.1 −538.3 −61.4 0.121 24hr  53.5 57.7 4.3 8.0 0.715

It will be obvious to those having skill in the art that many changesmay be made to the details of the above-described embodiments withoutdeparting from the underlying principles of the invention. The scope ofthe present invention should, therefore, be determined only by thefollowing claims.

1-64. (canceled)
 65. A method of preventing the onset of or treatingacute radiation syndrome, the method comprising: administering atherapeutically effective amount of a genistein formulation to a subjectthat has experienced exposure to radiation of at least 0.3 Gray or 30rads, the genistein formulation comprising: (i) nanoparticulategenistein; and (ii) a pharmaceutically acceptable suspension mediumcomprising an aqueous carrier, wherein the nanoparticulate genisteinexhibits a particle size distribution characterized by a D (0.50) of 0.5μm or less, and wherein the nanoparticulate genistein is present in theformulation at a concentration of between 250 mg/ml and 500 mg/ml. 66.The method of claim 65, wherein the genistein formulation is an oral orparenteral dosage form.
 67. The method of claim 66, wherein the oral orparenteral dosage form is selected from at least one of a capsule, agelatin capsule, a soft capsule, a liquid suspension, a pre-filledsachet, and a pre-metered dosing cup.
 68. The method of claim 65,wherein the nanoparticulate genistein exhibits a particle sizedistribution characterized by a D (0.50) of 0.2 μm or less.
 69. Themethod of claim 65, wherein the nanoparticulate genistein exhibits aparticle size distribution characterized by a D (0.50) of 0.2 μm or lessand a D (0.90) of 0.5 μm or less.
 70. The method of claim 65, whereinthe pharmaceutically acceptable suspension medium further comprises atleast one of a water soluble polymer and a nonionic surfactant.
 71. Themethod of claim 70, wherein the water soluble polymer is a polyvinylpyrrolidone (PVP), and wherein the water soluble polymer is present inthe formulation in an amount ranging from about 0.5% to about 15% (w/w).72. The method of claim 71, wherein the PVP is povidone K17.
 73. Themethod of claim 70, wherein the nonionic surfactant is one or more ofpolysorbate 80 and polysorbate 20, and wherein the nonionic surfactantis present in the formulation in an amount ranging from about 0.01% toabout 10% (w/w).
 74. A method of prophylactically treating exposure toradiation resulting from a therapeutic or diagnostic procedure, themethod comprising: administering a therapeutically effective amount of agenistein formulation to a subject prior to the therapeutic ordiagnostic procedure, the genistein formulation comprising: (i)nanoparticulate genistein; and (ii) a pharmaceutically acceptablesuspension medium comprising an aqueous carrier, wherein thenanoparticulate genistein exhibits a particle size distributioncharacterized by a D (0.50) of 0.5 μm or less, and wherein thenanoparticulate genistein is present in the formulation at aconcentration of between 250 mg/ml and 500 mg/ml.
 75. The method ofclaim 74, wherein the genistein formulation is an oral or parenteraldosage form.
 76. The method of claim 74, wherein the pharmaceuticallyacceptable suspension medium further comprises at least one of a watersoluble polymer and a nonionic surfactant.
 77. The method of claim 76,wherein the water soluble polymer is a polyvinyl pyrrolidone (PVP), andwherein the water soluble polymer is present in the formulation in anamount ranging from about 0.5% to about 15% (w/w).
 78. The method ofclaim 77, wherein the PVP is povidone K17.
 79. The method of claim 76,wherein the nonionic surfactant is one or more of polysorbate 80 andpolysorbate 20, and wherein the nonionic surfactant is present in theformulation in an amount ranging from about 0.01% to about 10% (w/w).80. A method of preventing the onset of or treating acute radiationsyndrome, the method comprising: administering a therapeuticallyeffective amount of a genistein formulation to a subject prior to thesubject being exposed to radiation greater than 0.3 Gray or 30 rads, thegenistein formulation comprising: (i) nanoparticulate genistein; and(ii) a pharmaceutically acceptable suspension medium comprising anaqueous carrier, wherein the nanoparticulate genistein exhibits aparticle size distribution characterized by a D (0.50) of 0.5 μm orless, and wherein the nanoparticulate genistein is present in theformulation at a concentration of between 250 mg/ml and 500 mg/ml. 81.The method of claim 80, wherein the pharmaceutically acceptablesuspension medium further comprises at least one of a water solublepolymer and a nonionic surfactant.
 82. The method of claim 81, whereinthe water soluble polymer is a polyvinyl pyrrolidone (PVP), and whereinthe water soluble polymer is present in the formulation in an amountranging from about 0.5% to about 15% (w/w).
 83. The method of claim 82,wherein the PVP is povidone K17.
 84. The method of claim 81, wherein thenonionic surfactant is one or more of polysorbate 80 and polysorbate 20,and wherein the nonionic surfactant is present in the formulation in anamount ranging from about 0.01% to about 10% (w/w).
 85. The method ofclaim 80, further comprising: continuing to administer therapeuticallyeffective amounts of the genistein formulation to the subject after thesubject has been exposed to radiation.